CN106566806B - The method that in vitro culture is enriched with CD8+T cells - Google Patents
The method that in vitro culture is enriched with CD8+T cells Download PDFInfo
- Publication number
- CN106566806B CN106566806B CN201610542602.9A CN201610542602A CN106566806B CN 106566806 B CN106566806 B CN 106566806B CN 201610542602 A CN201610542602 A CN 201610542602A CN 106566806 B CN106566806 B CN 106566806B
- Authority
- CN
- China
- Prior art keywords
- cell
- antibody
- cells
- culture
- minutes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/515—CD3, T-cell receptor complex
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to field of cell culture, and in particular to a kind of method of in vitro culture enrichment CD8+T cells, more particularly it relates to it is a kind of using stimulant from human peripheral blood mononuclear cell a large amount of directed expansion CD8+T cells method.T cell propagation quantity that technical scheme obtains is more, vigor is good, the ratio between CD8+T cells and CD4+T cells can be by about the 1 of normal physiological condition：2 improve to 2~5：1, while in purpose culture, based on total cell number, the content of central memory T cell is more than 30%.
Description
Technical field
The invention belongs to field of cell culture, and in particular to a kind of method of in vitro culture enrichment CD8+T cells, more specifically
Ground, the present invention relates to it is a kind of using stimulant from human peripheral blood mononuclear cell a large amount of directed expansion CD8+T cells side
Method.
Background technology
2011, cancer exceeded heart disease, became the global first big cause of death.WHO is announced in December, 2013, the whole world
Newly-increased cancer patient's number alreadys exceed 14,000,000 every year, this is compared with 12,700,000 people of statistical result of 2008, and number is significantly
Increase.The same period, the death toll of cancer patient also increased, and increase to 8,200,000 people from past 7,600,000 people.Report claims, and arrives
The year two thousand thirty, newly-increased cases of cancer will increase by 50%, reach annual 21600000 people.Immunity anticancer therapy is miscellaneous by Science within 2013
Will is chosen as first of annual 10 big technological breakthroughs.Since 2013, tumour cell and immunotherapy constantly obtain breakthrough,
Clinical research also achieves immense success, is the treatment means of current cancer therapies field most prospect, be expected to become after operation,
New conventional treatments after chemicotherapy method.
Cellular immunotherapy has been carried out extensively at present, is attempted in the LAK cell therapies of early stage, develops into CIK later
(Cytokine Induced Killer Cell), DC-CIK cell therapies etc..CIK cell is by IFN-γ, IL-2 and anti-
CD3 antibody (OKT3) breaks up after stimulating to be produced, wherein being added after when the 0th day IFN-γ for adding 1,000IU/ml, 24 is small
50ng/ml OKT3 and 300IU/ml IL-2, midfeather adds the fresh culture containing IL-2 in 2 days, afterwards by 2-3 weeks
Culture can obtain CIK cell.CIK cell mainly includes three subgroups, CD3+/CD56+NKT cells, CD3+/CD56-T cells and
CD3-/CD56+NK cells.NK cells play ADCC effects by non-specific surface's acceptor (such as Fc γ RIII, CD16) and identify
And killing tumor cell, the T cell by vitro culture can keep its TCR specific recognitions specificity target cell to play killing
Function, and CD3+/CD56+CIK cells are the main cell subgroups that CIK cell plays tumor-killing function, it passes through cell table
The MHC associated ligands A/B (MIC A/B) of the NKG2D molecular recognition tumor cell surfaces of face expression, is realized to the non-of tumour cell
The restricted killings of HLA.CD3+/CD56+CIK cells and the CD8+T cell phenotypes of terminal differentiation are closer to, it is considered to be from
CD8+T cell differentiations.Research at present thinks that CD3+/CD56+CIK cells are that tumor-killing effect is played in CIK cell
Main cell subgroup, therefore the important references index that its ratio is also accused frequently as CIK.The three classes cell subsets of CIK mainly leads to
The tumor-killing effect that non-specific killing ability reaches certain is crossed, as infantile tumour or tumor post-operation auxiliary treatment means,
In clinical studies certain contribution is made that for improving the life quality of patient.
Tumor-specific CTL cell is the direct effect cell of internal killing tumor cell, is known by the TCR of cell surface
The tumour antigen and HLA molecular complexes that other tumor cell surface presents, activate the intracellular series reactions of CTL, pass through secretion
The direct killing tumour cell such as perforin, granzyme.The new epitope that scientists study Tumor mutations produce in recent years is to machine
The influence of body antitumor action, is expected to promote the progress and clinical practice of tumor specific T cells treatment technology.Existing clinic is ground
Study carefully and show, tumour-specific is mutated at tumor tissues, the especially mutation of immunogenicity region antigen, can be produced new, swollen
Knurl specific antigen, these antigens can excite internal Specific T cell immunity to react, the T cell surface TCR affinity of generation
It is higher, so as to effectively play tumor cytotoxicity function.But immune tolerance environmental restrictions at tumor tissues specificity T is thin
Effective activation of born of the same parents and Function under vitro, it is necessary to implement human intervention, to mutant antigen specific T-cells progress body
Outer amplification and activation.
In T cell atomization, T cell experienceT cell, T effector cell, responsiveness memory t cell and
The different times of central memory T cell, the ability of its differentiation and proliferation is different, last eventually during immune cell therapy
The T cell of differentiation is since its differentiation and proliferation ability is relatively poor, and central memory T cell being capable of fast reaction and can be big
Amount propagation, the treatment for tumour etc. have more lasting action time, therefore, the maincenter produced in T cell incubation
Memory t cell divides group to be of great significance for later stage immune cell therapy effect.
In T cell atomization, its cell surface mark gradually changes, and shows as CD45RA and is gradually become by the positive
Feminine gender, and CD62L is gradually changed into positive from feminine gender, double staining analysis are shown as：Cell characterization is CD45RA+/CD62L
+；T effector cell is characterized as CD45RA+/CD62L-；Responsiveness memory t cell is characterized as CD45RA-/CD62L-；Maincenter
Memory t cell is characterized as CD45RA-/CD62L+.
CD8+T cells are presented by φt cell receptor (TCR) the specific recognition target cell surface of T cell surface expression
MHC-I/ epitope polypeptide complex molecules, by CD8 molecule combination target cell surface MHC-I molecules, raise CD3 molecules and will believe
The ITAM activation motifs of CD3 intracellulars number are transferred to, thus produce the first signal of t cell activation.Herein on basis, auxiliary swashs
Molecule CD28 living interacts with B7-1 equimoleculars, produces the secondary signal needed for t cell activation, produces cytotoxic T cell
(CTL) react.CD4+/CD8+T cell proportions are about CD4+ in fresh peripheral blood:CD8+=2:1.CD8+T cells are to play spy
The main cell subgroup of different in nature killing ability, and the method that there is no the external efficiently and directionally amplification CD8+T cells of maturation at present, because
This, the exploitation of external efficiently CD8+T cell directionals enrichment culture technique and reagent is for improving the oncotherapy based on CTL
It is of great significance, therefore clinically there is demand, the central memory T cell Sync enrichment particularly containing high level
The cultural method of CD8+T cells.
The content of the invention
To solve the above-mentioned problems, inventor provides a kind of side for being capable of efficient amplification CD8+T cells in vitro
Method.
It is an object of the present invention to provide a kind of method for the cell suspension for cultivating enrichment people's CD8+T cells, including with
Lower step：
Extract 5~100ml of people's whole blood and it is handled to obtain separated peripheral blood mononuclear cells (PBMCs)；
The separated PBMCs is sorted, obtains CD4+T cells and CD8+T cell T cell suspensions；
By the T cell suspension in 37 DEG C, 5%CO in Tissue Culture Plate2With under 100% damp condition with AIM-V
Serum-free cell culture medium carries out culture and obtains aim cell suspension in 7~14 days, wherein being added with respectively in the culture medium
The CD28 of the IL-2 of final concentration of 300U/ml, the CD3 antibody of final concentration of 5~10 μ g/ml and final concentration of 5~10 μ g/ml
Antibody；It is characterized in that the Tissue Culture Plate is the pre-coated Tissue Culture Plate for having CD3 antibody and CD28 antibody.
Preferably, the inoculum density of the T cell suspension is 5 × 105/ml。
It is preferably, described that pre-coated to have the Tissue Culture Plate of CD3 antibody and CD28 antibody realized by following methods
's：By the PBS buffer of the CD3 antibody containing final concentration of 5 μ g/ml and the CD28 antibody of final concentration of 5 μ g/ml according to 100 μ
L/ holes (96 orifice plate) or 500 μ l/ holes (24 orifice plate) are added to 4 DEG C of overnight incubations after Tissue Culture Plate.
Preferably, the processing is：The sterile phosphate buffer (PBS) that first peripheral blood of fresh collection is cooled down
One times of dilution, by diluted blood sample with 1：1~5 ratio is added in lymphocyte separation medium, at 25 DEG C with horizontal centrifuge
Centrifuged 20 minutes in 700g, second layer buffy coat is suctioned out into new sterile centrifugation tube, with isometric phosphate-buffered
Liquid in equal volume dilution after 25 DEG C of centrifugal forces 10 minutes with 800g；Supernatant is discarded, is resuspended with serum-free RPMI1640,
500g is centrifuged 5 minutes at 25 DEG C, discards supernatant, is resuspended, and it is clear to add the RPMI-1640 culture mediums containing 10% hyclone (FBS)
Wash, 500g is centrifuged 5 minutes at 25 DEG C, is resuspended after discarding supernatant with the RPMI-1640 culture mediums containing 10%FBS, is taken appropriate weight
Suspension in carrying out cell count on blood counting chamber, and with the RPMI-1640 culture mediums containing 10% serum by cell liquid adjust to
2.5×106The final densities of cell/ml.
Preferably, the PBS buffer is the PBS solution of 0.01M, pH 7.4.
Preferably, the sorting is realized by sorting column.
Preferably, the sorting is realized by the way that sorting column is placed in high-intensity magnetic field.
Preferably, the lymphocyte separation medium is purchased from Tianjin Hao oceans Bioisystech Co., Ltd.
Technical scheme obtains that T cell propagation quantity in cell suspension is more, vigor is good, CD8+T cells and CD4
The ratio between+T cell can be improved to 2: 1~5: 1 by about the 1: 2 of normal physiological condition, while in aim cell suspension, based on total thin
Born of the same parents' number, the content of central memory T cell is more than 30%.
Brief description of the drawings
Fig. 1 describes T cell separating effect flow cytometry.Wherein ordinate shows FITC mark CD8 antibody dyeing,
Abscissa shows the dyeing of perCP fluorescent marker CD4 antibody.
Fig. 2 describes two individual D1 and D2 blood samples and the 3rd day and the 6th day T cell subgroup point is cultivated after A, B, C processing
Analysis.
Fig. 3 describes two individual D1 and D2 blood samples and cultivates the 9th day and the 13rd day T cell subgroup after A, B, C processing
Analysis.
Fig. 4 describes two individual D1 and D2 blood samples and the 6th, 9,13 day T cell differentiation spy is cultivated after A, B, C processing
Sign.
Fig. 5 describes microscopy analysis in the 4th day after stimulant optimization and culture.
Embodiment
The present invention is by the specific embodiments and the drawings technical solution that the present invention is further explained, but the common skill in this area
Art personnel are understandable that：Detailed description below and embodiment are intended to illustrate the present invention, and should not be construed as to appoint
Where formula limitation is of the invention.It is well known to those skilled in the art, without departing from the spirit of the invention, the present invention can be done
Go out many modifications, such modification also falls into the scope of the present invention.
Following experimental methods are the experimental method of this area routine, used experiment material is such as unless otherwise specified
Without special instruction, the experiment material that can be readily obtained from commercial company is belonged to, antibody used in the present invention can be by
Commercial company is readily available.
T cell amplification in vitro culture is carried out first
Embodiment 1, volunteer's peripheral blood lymphocytes (PBMCs) separation：
Vein peripheral blood of the lymphocyte from individual used in the present invention.Individual by screening is through clinician's physical examination
After qualifications, detailed programs flow and the quantity of required blood are informed by experimenter, agrees to through volunteer and signs informed consent form,
By clinical worker, to volunteer, blood was collected.Final this project screening 2 healthy volunteers D1 and D2, when blood sampling, use
Contain EDTA-K2The 9ml disposal vacuums heparin tube (VACUETTE, Austrian Gray receive company) of anti-freezing, every volunteer adopts
Blood sample, is overturned anti-hemostasis-coagulation by blood about 20-25ml immediately after blood sampling.
1), phosphate buffer (0.01M PBS, the pH7.4, through 121 DEG C of height for first cooling down the peripheral blood of fresh collection
Pressure sterilizing) one times of dilution, by diluted blood sample with 1：1~5 ratio is added to preprepared 15ml separation of lymphocytes
In liquid (being purchased from Tianjin Hao oceans Bioisystech Co., Ltd), it need to be slowly added to chaotic to avoid interface；
2) the above-mentioned centrifuge tube containing lymphocyte separation medium and blood sample, is used into horizontal centrifuge under the conditions of 25 DEG C
(SORVALL Stratos, Thermo companies of the U.S.) 700g is centrifuged 20 minutes, and reduction of speed is adjusted to most slow during stopping；
Four layers of sample after centrifugation point, the superiors' blood plasma Pasteur pipette is suctioned out and is discarded, careful afterwards to suction out the
Two layers of buffy coat are thick pure PBMCs cells into new sterile centrifugation tube；
3) thick pure PBMCs cells, are diluted in equal volume with isometric phosphate buffer (0.01M PBS, pH7.4), it
Afterwards with the centrifugal force 10 minutes of 800g under the conditions of 25 DEG C；Supernatant is discarded, (U.S. GE is public with 7ml serum-frees RPMI1640
Take charge of Hyclone brands) it is resuspended, 500g is centrifuged 5 minutes at 25 DEG C.
Supernatant is discarded, is resuspended, adding 7ml, (FBS, U.S. Thermo Fisher company brands Australia are come containing 10% hyclone
Source) RPMI-1640 culture mediums (ibid, 1640 culture mediums are U.S.'s GE companies Hyclone brand cultures unless otherwise specified
Base) cleaning, 500g is centrifuged 5 minutes at 25 DEG C.
4) it is resuspended after, discarding supernatant with RPMI-1640 culture mediums of the 3ml containing 10%FBS, takes appropriate re-suspension liquid in hemocytometer
Cell count is carried out on number plate, and is adjusted cell liquid to 2.5 × 10 with the RPMI-1640 culture mediums containing 10% serum6Cell/
The final densities of ml, it is spare to obtain separated PBMCs cells.
Embodiment 2, T cell sorting
Sorting enrichment is carried out to the T lymphocytes in above-mentioned separated PBMCs cells by immunomagnetic beads method, to obtain height
The T cell of purity, to carry out follow-up cultivation.
1) first by the separated PBMCs cells of above-mentioned acquisition according to every 1 × 107The CD4 antibody couplings of cell and 20 μ l
The CD8 antibody-coupled magnetic beads of magnetic bead and 20 μ l (being purchased from Mei Tian Ni Bioisystech Co., Ltd of Germany) are mixed, 4 DEG C of postposition
Be incubated 15 minutes, with containing 5% hyclone (FBS, U.S. Thermo Fisher company brands Australia source) and 2mM
The PBS buffer (PBS-F buffer solutions) of EDTA-Na is cleaned twice, and 500g is centrifuged 5 minutes at 25 DEG C.
2) above-mentioned cell is resuspended with 500 μ l PBS-F buffer solutions, and by the disposable sterilized strainer of 200 mesh (purchased from Germany
Mei Tian Ni Bioisystech Co., Ltd) filtering, the impurity in cell suspension is removed, so that cell can pass through sorting column
(MS sorts column, purchased from German Mei Tian Ni Bioisystech Co., Ltd), it is thin to obtain the PBMCs that CD4 and CD8 antibody magnetic bead combines
Born of the same parents.
3) high-intensity magnetic field (MiniMACS will be placed on purchased from the MS of German Mei Tian Ni Bioisystech Co., Ltd sorting columns
Separator, purchased from German Mei Tian Ni Bioisystech Co., Ltd) in, with 0.5ml PBS-F buffer solutions rinse 2 times, to last
After rinse PBS-F buffer solution flows completely out sorting column, it is spare to obtain processed MS sortings column.
4) by above-mentioned steps 2) obtain CD4 and CD8 antibody magnetic bead combine PBMCs cells be slowly added into step 3) place
MS sorting columns after reason, combining the T cell of CD4 antibody magnetic bead and CD8 antibody magnetic beads at this time will be detained under magnetic fields
In MS sorts column, and other cells of uncombined magnetic bead as flow through cell then along sorting column outflow.Delayed with the PBS-F of 1ml
Fliud flushing is slowly added into sorting column in three times, and the cell for making to be not associated with magnetic bead in sorting column elutes sorting column completely, to obtain
Obtain the T cell for combining CD4 antibody magnetic bead and CD8 antibody magnetic beads of higher purity.
5) column will be sorted away from magnetic field containing the T cell for combining CD4 antibody magnetic bead and CD8 antibody magnetic beads, add 1ml's
PBS-F buffer solutions, the CD4/CD8T cells that are combined with magnetic bead in sorting column are released with MS sorting column pistons, 1500rpm/ minutes from
The heart 5 minutes, discard after supernatant with AIM-V serum-free cell culture mediums (Medium-CTS, similarly hereinafter) clear
Wash twice, and with white containing 10% people AB serum (v/v) (being purchased from Jiangsu En Moasai Bioisystech Co., Ltd) and 300IU/ml
The AIM-V culture mediums (source is same as above) of cytokine -2 (IL-2) (being purchased from PeproTech companies) be resuspended count up to concentration for 5 ×
105The cell suspension of cell/ml, cell suspension is added in 24 porocyte culture plates, in 37 DEG C, 5%CO2With 100% humidity
Under the conditions of cultivated, obtain the high-purity T cell based on CD4+ and CD8+T cells, in case subsequent stimuli handle.
6) during magnetic bead sorting, above-mentioned steps 5 are taken) obtain concentration be 1 × 105CD4+ and CD8+T cell suspensions,
CD4 and CD8 fluorescent antibody stainings are carried out respectively, are evaluated separating effect (Fig. 1) with flow cytometry (FACS) afterwards.
By the T cell sorting and flow cytometer showed verification to two healthy individuals D1 and D2, CD4+T cells are in D1 and D2 after finding sorting
Middle accounting 57.5% and 71.9%, CD8+T cell accounting respectively are respectively 31.2% and 19.4%.Sub-elected from D1 and D2
CD4+ and CD8+T cells add up to that to account for total lymphocyte ratio be respectively 88.7% and 91.3%.
Embodiment 3, the amplification of T cell stimulated in vitro
1) by embodiment 2 step 5) obtain the high-purity T cell based on CD4+ and CD8+T cells carry out respectively with
Lower tri- kinds of processing modes of A, B, C:
A. the magnetic bead of CD3 and CD28 antibody couplings is added；
B. it is placed in the pre-coated Tissue Culture Plate for having CD3 antibody and CD28 antibody；
C. CD3 the and CD28 antibody of solution state is added；
To compare the T cell that T cell propagation is horizontal and expands under the conditions of different stimulated combining form and training method
Subgroup and level of differentiation.Specific addition or condition of culture are as follows：
The magnetic bead culture of A.CD3 and CD28 antibody couplings：The magnetic bead for being coated with CD3 and CD28 antibody (is purchased from Life
Technology companiesHuman T-Activator CD3/CD28) according to magnetic bead：T cell=1：1 quantity
Ratio is taken out and adds the AIM-V culture mediums of 1ml, in high-intensity magnetic field (MiniMACS Separator, purchased from Germany U.S. day Ni biologies
Technology Co., Ltd.) under the conditions of discard supernatant.This step is repeated afterwards 1 time, be resuspended with the AIM-V culture mediums of 50-100 μ l volumes
It is spare；By above-mentioned steps 5) in the high-purity T cell based on CD4+ and CD8+T cells that sub-elects according to 5 × 105/ ml is close
Degree, which is added in 24 porocyte culture plates of the antibody-coupled magnetic beads containing CD3 and CD28, to be cultivated, and is that CD3 and CD28 antibody is even
The magnetic bead culture T cell of connection；
B. it is placed in the Tissue Culture Plate culture of pre-coated CD3 and CD28 antibody：Prepare above-mentioned steps 5) obtain with
CD4+ and CD8+T day before cell, by CD3 antibody, (clone number is OKT-3, grade purified (the Functional Grade of feature
Purified), concentration 1mg/ml, purchased from eBioscience companies) and CD28 antibody (clone number is CD28.2, feature etc.
Level purifying (Functional Grade Purified), concentration 1mg/ml, purchased from eBioscience companies), respectively with end
5 μ g/ml of concentration are diluted in a PBS buffer, and are added according to 100 μ l/ holes (96 orifice plate) or 500 μ l/ holes (24 orifice plate)
4 DEG C of overnight incubations afterwards；Next day, discard supernatant solution in hole, and by above-mentioned steps 5) in sub-elect with CD4+ and CD8+T cells
Based on high-purity T cell according to 5 × 105/ ml density is added in the pre-coated orifice plate for having CD3 and CD28 antibody and is trained
Support, be the Tissue Culture Plate culture T cell of pre-coated CD3 and CD28 antibody；
C. solution state CD3 and CD28 antibody culture is added：By above-mentioned steps 5) in sub-elect it is thin with CD4+ and CD8+T
High-purity T cell based on born of the same parents is according to 5 × 105/ ml density adds 24 orifice plates, and per hole 1ml, it is respectively 10 μ g/ to add final concentration
CD3 the and CD28 antibody of ml is cultivated, and is solution state CD3 and CD28 antibody culture T cell.
The T cell handled respectively by above-mentioned A, B and C is cultivated under 37 DEG C, 5%CO2 and 100% damp condition
14 days.The the 3rd, 6,9,13 day after A, B and C processing is carried out respectively, the T cell cultivated is counted respectively, and according to
Cell growth status is counted again, according to 5 × 105The density of/ml readjusts cell density, and with containing comparable sodium
A, the culture medium of stimulant mentioned in B and C processing and combinations thereof is supplemented to 1ml volumes.Each stimulation group takes no less than 1 ×
105Cell in case the detection of follow-up flow cytometry, to stimulating the T cell quantity produced, T cell subgroup and memory differentiation special
Sign etc. carries out qualitative and quantitative detection.
Embodiment 4, CD8+T cell high-efficient amplification in vitro cultures
The cultural method provided according to embodiment 3, separately sampled is flowed for the 3rd, 6,9 and 13 day after T cell culture
Formula cell detection, T cell subgroup and differentiating characteristic after being cultivated with evaluation.
1) CD4/CD8T cell subsets is analyzed
Take T cell 1 × 10 after cultivating5It is a, centrifuged 5 minutes with 1500rpm/ minutes, delayed after discarding supernatant with the PBS of 1ml
Fliud flushing carries out 2 eccentric cleanings；Last time is resuspended after centrifuging with 50 μ l PBS, is added PE-anti-CD4 antibody and (is purchased from BD
Biosciences) and FITC-anti-CD8 antibody (being purchased from BD Biosciences) each 1 reacting dose, it is incubated at room temperature 15 points
Clock, afterwards with the PBS buffer eccentric cleaning of 1ml twice, centrifuges 5 minutes for 1500rpm/ minutes.
Last time is resuspended after centrifuging with 500 μ l PBS buffer, is added streaming pipe and (is purchased from BD
Biosciences upper machine testing (BD FACSCalibur double excitations stream type cell analyzer), the data obtained warp are prepared in)
FlowJo 7.6.1 softwares (FLOWJO, LLC company) are analyzed.
The result of table 1, individual D1 and D2 after A is handled (unit accounts for the percentage of all cells for the cell)
The result of table 2, individual D1 and D2 after B is handled (unit accounts for the percentage of all cells for the cell)
The result of table 3, individual D1 and D2 after C is handled (unit accounts for the percentage of all cells for the cell)
CD8 when the 3rd day after A, B and C processing culture:The higher feelings of CD4 ratios are still presented in cd4 t cell ratio
Condition, passes through the detection to two healthy individuals of D1 and D2, the magnetic bead culture T cell of discovery D1 individual CD3 and CD28 antibody couplings
The Tissue Culture Plate culture T cell (Coated in Fig. 2 of (CD3/CD28beads groups in Fig. 2), pre-coated CD3 and CD28 antibody
CD3/CD28Ab groups) and solution state CD3/CD28 antibody cultures T cell (Soluble CD3/CD28Ab groups in Fig. 2) in CD4+
T cell/CD8+T cell proportions are respectively：25.0%/65.1%, 21.5%/67.4%, 18.2%/75.8%.In D2 individuals
The magnetic bead culture T cell (CD3/CD28beads groups in Fig. 2) of middle CD3 and CD28 antibody couplings, pre-coated CD3 and CD28 antibody
Tissue Culture Plate culture T cell (Coated CD3/CD28Ab groups in Fig. 2) and solution state CD3/CD28 antibody cultures T it is thin
CD8+T cells/CD4+T cell proportions are respectively in born of the same parents' (Soluble CD3/CD28Ab groups in Fig. 2)：23%/63.5%,
12.4%/84.2%, 7.57%/87.5%.
2) after the 6th day original ratio is maintained with CD4/CD8T cells in the T cell of CD3/CD28 antibody magnetic beads after incubation
Example, and CD4/CD8T cell proportions are opened in the T cell culture of pre-coated CD3/CD28 antibody and solvable CD3/CD28 antibody culture
Beginning takes a turn for the worse.The magnetic bead culture T cell (CD3/CD28beads groups in Fig. 2) of D1 individual CD3 and CD28 antibody couplings, pre- bag
By the Tissue Culture Plate culture T cell (Coated CD3/CD28Ab groups in Fig. 2) and solution state CD3/ of CD3 and CD28 antibody
CD8+T cells/CD4+T cell proportions are respectively in CD28 antibody cultures T cell (Soluble CD3/CD28Ab groups in Fig. 2)：
20.9%/70.5%, 62.4%/30.8%, 56.1%/36.2%.The magnetic bead training of CD3 and CD28 antibody couplings in D2 individuals
T cell (CD3/CD28beads groups in Fig. 2), the Tissue Culture Plate culture T cell of pre-coated CD3 and CD28 antibody are supported (in Fig. 2
Coated CD3/CD28Ab groups) and solution state CD3/CD28 antibody cultures T cell (Soluble CD3/CD28Ab in Fig. 2
Group) in CD8+T cells/CD4+T cell proportions be respectively：23.1%/70.5%, 59.4%/19.4%, 53.9%/33.6%.
3) still maintained substantially with CD4/CD8T cells in the T cell of CD3/CD28 antibody magnetic beads after the 9th day after incubation
Original scale, and CD4/CD8T cells in the T cell culture of pre-coated CD3/CD28 antibody and solvable CD3/CD28 antibody culture
Ratio, which reverses, to be continued.It is the magnetic bead culture T cell (CD3/CD28beads groups in Fig. 3) of D1 individual CD3 and CD28 antibody couplings, pre-
It is coated with the Tissue Culture Plate culture T cell (Coated CD3/CD28Ab groups in Fig. 3) and solution state of CD3 and CD28 antibody
CD8+T cells/CD4+T cell proportions difference in CD3/CD28 antibody cultures T cell (Soluble CD3/CD28Ab groups in Fig. 3)
For：19.1%/73.4%, 58.8%/31%, 51.9%/36.2%.The magnetic bead of CD3 and CD28 antibody couplings in D2 individuals
Cultivate T cell (CD3/CD28beads groups in Fig. 3), Tissue Culture Plate culture T cell (Fig. 3 of pre-coated CD3 and CD28 antibody
Middle Coated CD3/CD28Ab groups) and solution state CD3/CD28 antibody cultures T cell (Soluble CD3/CD28Ab in Fig. 3
Group) in CD8+T cells/CD4+T cell proportions be respectively：18%/75.3%, 70.5%/13.9%, 53.5%/28.2%.
Still maintained substantially with CD4/CD8T cells in the T cell of CD3/CD28 antibody magnetic beads after the 13rd day after incubation
Original scale, and pre-coated CD3/CD28 antibody realizes further improving for CD8+T cell proportions, and solvable CD3/CD28 resists
CD4/CD8T cell proportions reverse lasting maintain in the T cell culture of body culture.The magnetic bead of D1 individual CD3 and CD28 antibody couplings
Cultivate T cell (CD3/CD28beads groups in Fig. 3), Tissue Culture Plate culture T cell (Fig. 3 of pre-coated CD3 and CD28 antibody
Middle Coated CD3/CD28Ab groups) and solution state CD3/CD28 antibody cultures T cell (Soluble CD3/CD28Ab in Fig. 3
Group) in CD8+T cells/CD4+T cell proportions be respectively：17.8%/77.6%, 63.4%/30.7%, 62.6%/30.4%.
In D2 individuals the magnetic bead culture T cell (CD3/CD28beads groups in Fig. 3) of CD3 and CD28 antibody couplings, pre-coated CD3 and
The Tissue Culture Plate culture T cell (Coated CD3/CD28Ab groups in Fig. 3) and solution state CD3/CD28 antibody of CD28 antibody
CD8+T cells/CD4+T cell proportions are respectively in culture T cell (Soluble CD3/CD28Ab groups in Fig. 3)：15.4%/
80%, 83.4%/6.96%, 52.3%/31.8%.
The 5. external high-efficient culture of responsiveness memory T cell of embodiment：
1) T cell differentiating characteristic is analyzed
The cell of the 3rd, 6,9,13 day is through 1 × 10 after taking A, B, C and stimulating culture5, 1500rpm/min centrifugation 5min, discard
After supernatant 2 eccentric cleanings are carried out with the PBS buffer of 1ml；Last time is resuspended after centrifuging with 50 μ l PBS, adds PerCP-
Anti-CD45RA antibody and APC-anti-CD62L antibody (being purchased from BD Bioscience companies) each 1 reacting dose, room temperature
It is incubated 15 minutes, afterwards with 1ml PBS eccentric cleanings twice, is centrifuged 5 minutes with 1500rpm/ minutes rotating speeds.
Last time is resuspended after centrifuging with 500 μ l PBS buffer, obtains memory phenotype cell sample to be detected, and
It is added into streaming pipe and prepares upper machine testing (BD FACSCalibur double excitations stream type cell analyzer).
2) interpretation of result
In the present embodiment, three kinds of different disposals to embodiment 2 and the T cell of culture to the 6th, 9 and 13 day are broken up sub-
Group is detected.The result shows that (Fig. 4), central memory T cell subset proportions are in two Healthy Peoples of D1 and D2 with culture
The extension of time is in the trend increased.In D1 samples, pre-coated CD3/CD28 antibody culture and the training of solvable CD3/CD28 antibody
Foster central memory T cell (fourth quadrant) was respectively reached in the ratio of the 6th, 9 and 13 day：10%th, 38.8% and
51.4% (B groups)；7.22%th, 42.3% and 37.5% (C groups), and the central memory T cell ratio that A tissue cultures are supported is the 6th, 9
Ratio with 13 days respectively reaches 1.23%, 30% and 58.8%.And on the other hand, the processing culture of A groupsT cell
Ratio after incubation still reach within the 13rd day 26.7%, and what B groups processing culture and the processing of C groups were cultivatedT cell ratio is only
For 2.19% and 9.26%.
In D2 samples, the central memory T cell (fourth quadrant) of B groups processing culture and the processing culture of C groups is the 6th, 9
Ratio with 13 days respectively reaches：10%th, 46.8% and 38.9% (B groups)；7.22%th, 56.8% and 38.1% (C groups), and
The central memory T cell ratio of A groups processing culture respectively reached 1.23%, 64.2% and in the ratio of the 6th, 9 and 13 day
63.8%.And on the other hand, the processing culture of A groupsThe ratio of T cell after incubation still reach 14.5% within the 13rd day,
And what B groups and the processing of C groups were cultivatedT cell ratio is only 1.26% and 6.43%.
Therefore, find through this embodiment, memory-type is presented in the T cell produced under three kinds of different culture incentive conditions
The increased phenomenon of T cell ratio.Analysis to the T cell differentiation subgroup of three kinds of different stimulated CMC models finds that the processing of A groups is trained
Foster T cell can differentiate more central memory T cells and higher proportion of at the 13rd dayT cell, and B groups
The T cell of processing culture and the processing culture of C groups has higher proportion of T effector cell and responsiveness memory t cell.
Embodiment 6.CD3 and CD28 antibody are coated with concentration and stimulant Combinatorial Optimization
By embodiment 4 it can be found that pre-coated CD3/CD28 antibody can produce higher proportion of CD8+T cells and
Central memory T cell, this case have further carried out further optimization to incentive condition and stimulant combination.
The present embodiment is separately added into T cell culture hole is originated：
A. culture medium blank control：AIM-V serum-free cell culture mediums；
B.IL-2 (300U/ml)+AIM-V serum-free cell culture mediums；
C.IL-2 (300U/ml)+anti-CD3 (10ug/ml)+AIM-V serum-free cell culture mediums；
D.IL-2 (300U/ml)+anti-CD28 (10 μ g/ml)+AIM-V serum-free cell culture mediums；
E.IL-2 (300U/ml)+anti-CD3 (10 μ g/ml)+anti-CD28 (10 μ g/ml)+AIM-V serum-free cells
Culture medium；
F.IL-2 (300U/ml)+anti-CD3 (5 μ g/ml)+anti-CD28 (5 μ g/ml)+AIM-V serum-free cells are trained
Support base
Carry out micro- Microscopic observation within the 4th day after incubation, it is in disperse to find the culture hole T cell without any stimulation, agglomerating
Few, growth conditions are poor；The culture hole of addition IL-2 has agglomerating T cell to occur, and the culture hole for being coated with CD3 or CD28 antibody goes out
Existing more T cell group, while the culture hole cell mass for being coated with CD3 and CD28 antibody is significantly more than a kind of single training of antibody of coating
Hole is supported, cell suspension counting is extracted and finds that cell suspension counting can reach 1 × 109More than a, and 5 μ g/ml and 10 μ g/ml two
It is not significantly different between a coating concentration.
Required height can be obtained with 5 μ g/ml coating CD3 and CD28 antibody culture T cells it can be seen from description more than
Ratio CD8+T cells, available for clinical treatment.
Claims (5)
1. a kind of method of in vitro culture enrichment people's CD8+T cells, comprises the following steps：
(1) extract 5~100ml of people's whole blood and it is handled to obtain separated peripheral blood mononuclear cells；
(2) the separated peripheral blood mononuclear cells is sorted, obtains CD4+T cells: CD8+T cells ratios are 1：
1~1：5 T cell suspension；
(3) the T cell suspension is inoculated into the AIM-V serum-free cell culture mediums in Tissue Culture Plate, 37 DEG C,
7~14 days acquisition aim cell suspensions are cultivated under 5%CO2 and 100% damp condition, wherein also adding respectively in the culture medium
The CD3 antibody of IL-2, final concentration of 5~10 μ g/ml added with final concentration of 300U/ml and final concentration of 5~10 μ g/ml's
CD28 antibody；
It is characterized in that, the Tissue Culture Plate is the pre-coated Tissue Culture Plate for having CD3 antibody and CD28 antibody, it is described
It is pre-coated that to have the Tissue Culture Plate of CD3 antibody and CD28 antibody prepared by following methods：
By the PBS buffer of the CD3 antibody containing final concentration of 5 μ g/ml and the CD28 antibody of final concentration of 5 μ g/ml according to right
4 DEG C of overnight incubations after 96 orifice plates are added to Tissue Culture Plate with 100 μ l/ holes or for 24 orifice plates with the amount in 500 μ l/ holes.
2. according to the method described in claim 1, it is characterized in that, the inoculum density of the T cell suspension described in step (3) is
5×105Cell/ml.
3. according to the method described in claim 1, it is characterized in that, the processing described in step (1) is：
The sterile phosphate buffer that first peripheral blood of fresh collection is cooled down dilutes one times, by diluted blood sample with 1：1~
1：5 ratio is added in lymphocyte separation medium, is centrifuged 20 minutes in 700g with horizontal centrifuge at 25 DEG C, is suctioned out the second layer
Buffy coat is diluted after 25 DEG C of centrifugal force with 800g into new sterile centrifugation tube with isometric phosphate buffer
Centrifugation 10 minutes；Supernatant is discarded, is resuspended with serum-free RPMI1640,500g is centrifuged 5 minutes at 25 DEG C, discards supernatant, is resuspended,
Add the RPMI-1640 culture mediums containing 10% hyclone to clean, 500g is centrifuged 5 minutes at 25 DEG C, is discarded after supernatant with containing
The RPMI-1640 culture mediums of 10%FBS are resuspended, and take appropriate re-suspension liquid in carrying out cell count on blood counting chamber, and with containing
The RPMI-1640 culture mediums of 10% serum adjust cell liquid to 2.5 × 106The final densities of cell/ml.
4. according to the method described in claim 1, it is characterized in that, the sorting described in step (2) is realized by sorting column
's.
5. according to the method described in claim 4, it is characterized in that, the sorting is by the way that sorting column is placed on high-intensity magnetic field
Middle realization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610542602.9A CN106566806B (en) | 2016-07-11 | 2016-07-11 | The method that in vitro culture is enriched with CD8+T cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610542602.9A CN106566806B (en) | 2016-07-11 | 2016-07-11 | The method that in vitro culture is enriched with CD8+T cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106566806A CN106566806A (en) | 2017-04-19 |
| CN106566806B true CN106566806B (en) | 2018-05-04 |
Family
ID=58532195
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610542602.9A Active CN106566806B (en) | 2016-07-11 | 2016-07-11 | The method that in vitro culture is enriched with CD8+T cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106566806B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603948B (en) * | 2017-08-14 | 2019-07-05 | 武汉圣济科技有限公司 | A method of direct external evoked human peripheral blood single nucleus cell becomes compound antigen-non-specific regulatory T cells |
| CN108315298B (en) * | 2018-02-07 | 2020-10-20 | 安徽古一生物科技有限公司 | Gamma T cell culture method |
| CN108588022B (en) * | 2018-05-10 | 2019-12-10 | 英威福赛生物技术有限公司 | Method for enriching human CD4+ and CD8+ TCM cells through in vitro culture |
| CN108441473B (en) * | 2018-05-10 | 2020-02-07 | 高山 | Method for enriching CD8+ T cells in vitro |
| CN108841789A (en) * | 2018-06-19 | 2018-11-20 | 中国人民解放军陆军军医大学第二附属医院 | A kind of method of Activated in Vitro mouse T lymphocyte |
| CN109486761A (en) * | 2019-01-17 | 2019-03-19 | 汇麟生物科技(北京)有限公司 | The cultural method of peripheral blood CTL cell |
| CN111621477B (en) * | 2020-05-25 | 2021-06-22 | 合源生物科技(天津)有限公司 | T cell sorting method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618498A (en) * | 2012-03-26 | 2012-08-01 | 时宏珍 | Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte) |
| CN102839153A (en) * | 2012-09-13 | 2012-12-26 | 济南泰生生物技术有限公司 | Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major |
| CN104651311A (en) * | 2014-09-03 | 2015-05-27 | 深圳市茵冠生物科技有限公司 | Kit for preparing DC-CTL and application of kit |
| CN105543170A (en) * | 2015-12-31 | 2016-05-04 | 中山大学 | Composition capable of stimulating expansion of T cells |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1241249A1 (en) * | 2001-03-12 | 2002-09-18 | Gerold Schuler | CD4+CD25+regulatory T cells from human blood |
-
2016
- 2016-07-11 CN CN201610542602.9A patent/CN106566806B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618498A (en) * | 2012-03-26 | 2012-08-01 | 时宏珍 | Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte) |
| CN102839153A (en) * | 2012-09-13 | 2012-12-26 | 济南泰生生物技术有限公司 | Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major |
| CN104651311A (en) * | 2014-09-03 | 2015-05-27 | 深圳市茵冠生物科技有限公司 | Kit for preparing DC-CTL and application of kit |
| CN105543170A (en) * | 2015-12-31 | 2016-05-04 | 中山大学 | Composition capable of stimulating expansion of T cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106566806A (en) | 2017-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106566806B (en) | The method that in vitro culture is enriched with CD8+T cells | |
| CN102268405B (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| CN104357394B (en) | Culture method of autologous peripheral blood lymphocyte DC-CIK (Dendritic Cell- Cytokine-induced Killer) | |
| CN108588022B (en) | Method for enriching human CD4+ and CD8+ TCM cells through in vitro culture | |
| CN107083360B (en) | A kind of method of external evoked amplification human antigen's non-specificity regulatory T cells | |
| CN106434554B (en) | The preparation method of NK cell | |
| CN109666640A (en) | The method of the external pure culture of natural killer cells | |
| US20150210982A1 (en) | Isolation and Use of Human Regulatory T Cells | |
| EP3980777A1 (en) | Methods for manufacturing t cells by direct sorting and compositions thereof | |
| CN106011061A (en) | In-vitro large-scale amplification method of natural killer cells | |
| CN107365748A (en) | The memory immune cell of PMNC induction and application | |
| CN112251406A (en) | Exosome sorting method for NK cell activation stage | |
| CN117106846A (en) | Method for evaluating immune regulation function of stem cell preparation with high reliability | |
| CN109207427A (en) | A method of hematopoietic progenitor cells are changed into candidate stem cell | |
| WO2015014291A1 (en) | Lymph cell amplification and activation method via serum-free cultivation | |
| CN105296421B (en) | The T cell and preparation method of a kind of activation of bispecific antibody and application | |
| CN112852728B (en) | LCL-NK cell combined culture method based on peripheral blood, cell and product | |
| CN104762261A (en) | Tumor infiltrating lymphocytes separation method | |
| CN105969731B (en) | A method of High Fragmentation activity til cell is largely prepared using pernicious Pleural effusions | |
| CN108441481A (en) | A kind of Chimeric antigen receptor T cell and its cultural method | |
| CN108690829B (en) | Method for efficiently amplifying NK cells | |
| CN106222139B (en) | A method of High Fragmentation activity til cell is largely prepared using concretionary pernicious Pleural effusions | |
| CN108795858A (en) | The screening technique of high anti-cancer activity T cell and application | |
| CN108441473A (en) | A kind of method of ex vivo enrichment CD8+* T cells | |
| CN103160464A (en) | Preparation method and kit for effector lymphocyte capable of rapidly proliferating, kit and application of kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |